The long-term goal of this project is to understand the genetic and molecular mechanisms underlying phenotypic evolution. This includes discovering the genes responsible for phenotypic divergence, identifying which changes within these genes affect the phenotype, and determining how these changes alter molecular, cellular, and developmental processes. Preliminary studies using a combination of genetic mapping and transgenic analysis show that pigmentation differences between two closely related Drosophila species, D. americana and D. novamexicana, are caused (in part) by divergence within the tan gene. tan encodes an enzyme that catalyzes the conversion of N-beta-alanyl-dopamine into dopamine, which is a necessary step for the production of dopamine melanin. Fine-scale genetic mapping identified a region of tan containing only non-coding differences, which suggests that divergent tan expression between species contributes to pigmentation divergence, and greater abundance of tan mRNA was observed in D. americana than D. novamexicana. The goal of the proposed project is to locate all divergent sites within tan that affect pigmentation and to determine how they alter pigmentation development. Specifically, the aims of this proposal are to: (1) identify nucleotide differences between species-specific tan alleles that affect pigmentation, (2) determine the phenotypic consequences of these changes in D. novamexicana and D. americana, and (3) determine how these sites affect tan mRNA and protein expression. Transgenic approaches used in this project allow direct comparisons between genotypes that differ for as little as one nucleotide, and the analysis of transgenes in multiple species allows cis- and trans-regulatory changes responsible for tan divergence to be resolved. When completed, this project will improve our general understanding of the genetic changes and molecular mechanisms underlying phenotypic differences in complex traits.